KR101497506B1 - The Pharmaceutical composition and functional food for inhibiting angiotensin-I converting enzyme - Google Patents

Abstract

The present invention provides an angiotensin-converting enzyme (ACE) inhibitor and a method for producing the same, which comprises an extract of Alaska pollack or an enzyme hydrolyzate of Alaska pollack as an active ingredient. In addition, it provides a structure of a peptide having an angiotensin converting enzyme inhibitory activity. The pharmaceutical composition for inhibiting angiotensin converting enzyme according to the present invention and the health functional food can be used for prevention and treatment of hypertension.

Description

[0001] The present invention relates to a pharmaceutical composition for inhibiting angiotensin-converting enzyme and a functional food for inhibiting angiotensin-

The present invention relates to a pharmaceutical composition for inhibiting angiotensin converting enzyme and a health functional food useful for prevention and treatment of hypertension. More specifically, the present invention relates to a substance having an angiotensin converting enzyme inhibitory action and a composition for the peptide thereof.

Hypertension is a chronic degenerative disease of the circulatory system and is explained by the physiological mechanism of the renin-angiotensin system, which plays an important role in regulating blood pressure and electrolyte balance. As shown in Fig. 1, the angiotensin converting enzyme (ACE) is an angiotensin converting enzyme (ACE), which cleaves a dipeptide (His-Leu) from angiotensin I, which is a decapeptide, Ⅱ. The increase in angiotensin II produced by angiotensin converting enzyme promotes hypertension by promoting strong hypertension and secretion of aldosterone, an antidiuretic hormone, and inhibiting the excretion of water and sodium to increase circulating blood. In addition, angiotensin converting enzyme degrades and inactivates vascular relaxant bradykinin, resulting in a rise in blood pressure. Therefore, inhibition of the activity of angiotensin converting enzyme inhibits vasoconstriction and blood pressure lowering effect can be shown to lower the hypertension.

Antihypertensive agents for hypertension include diuretics, beta-blockers, sympathomimetics, vasodilators, Ca-antagonists and AT ₁ -reptor blockers such as candasatan, ibesartan, losartan, and the like. However, most of these drugs cause side effects such as elevated blood viscosity, electrolyte imbalance, diabetes induction, worse heart failure, dysfunction, and kidney dysfunction.

On the other hand, as interest in health and longevity has been concentrated in recent years, various physiological activities such as anticancer, anti-aging and anti-hypertension, which are represented by food ingredients, are being carried out variously. Substances derived from food are of great value in terms of their natural nature.

Particularly, among peptides derived from foods, peptides are intermediate products of the decomposition process of proteins into amino acids, and generally have a molecular weight of less than 10,000 dalton and have various physiological activities. Peptides originated from food proteins do not have functional properties in the original protein structure, but hydrolysis with proteases has the advantage of being able to produce and modify by gene manipulation, and to expect consumer taste and food safety. In many studies, peptide components have been reported to exhibit a variety of physiological activities including immune enhancement, calcium absorption enhancement, antithrombotic, cholesterol regulation, and antihypertension.

Therefore, the pharmaceutical composition for inhibiting angiotensin converting enzyme derived from food is of great value in that these components are natural products. Therefore, there is a continuing research on the search for a natural substance having a strong angiotensin converting enzyme inhibitory activity, and it is necessary to study a novel food-derived substance having a high angiotensin converting enzyme inhibitory activity. In addition, it is necessary to analyze the structure of the peptide having an angiotensin converting enzyme-inhibiting activity by separating and purifying an angiotensin converting enzyme-inhibiting substance derived from food.

Accordingly, a problem to be solved by the present invention is to provide a pharmaceutical composition for inhibiting angiotensin converting enzyme from food, a health functional food, and a method for producing the same.

Yet another object of the present invention is to provide a structure of a peptide having an angiotensin converting enzyme inhibitory activity from food.

In order to solve the above-mentioned problems, the present invention relates to an angiotensin converting enzyme (ACE) inhibitor, preferably an angiotensin converting enzyme (ACE) inhibitor, Compositions and health functional foods. That is, the present invention provides a medicinal use of an enzyme hydrolyzate of an alopecia extract or alfalfa.

Also, the present invention provides a method for producing a recombinant yeast strain, comprising the steps of: (S1) adding a protease to a keyhole; (S2) heating the reaction solution obtained in the step S1 to deactivate the proteolytic enzyme in the reaction solution; And (S3) a step of filtering the supernatant obtained by centrifuging the reaction solution obtained in the step (S2). The method for producing an enzyme hydrolyzate of angiotensin converting enzyme inhibitor according to claim 1,

Also, the present invention provides a method for producing a recombinant yeast strain, comprising the steps of: (S1) adding a protease to a keyhole; (S2) heating the reaction solution obtained in the step S1 to deactivate the proteolytic enzyme in the reaction solution; And (S3) centrifuging the reaction solution obtained in the step (S2) to filter the supernatant. The method for enhancing angiotensin converting enzyme inhibitory activity of the present invention is thus provided.

The present invention provides a pharmaceutical composition for inhibiting angiotensin converting enzyme, preferably a pharmaceutical composition for lowering blood pressure, and a health functional food, which comprises a peptide comprising Arg-Gln-Pro as an active ingredient. That is, the present invention provides a medicament for inhibiting angiotensin converting enzyme of a peptide comprising Arg-Gln-Pro.

In the present invention, the health functional food refers to all foods prepared or processed by using raw materials or ingredients having useful functionality, including, for example, health supplements, functional foods, nutritional supplements and adjuvants.

As shown in FIG. 1, hypertension is explained by the physiological mechanism of the renin-angiotensin system. Inhibition of angiotensin converting enzyme shown in FIG. 1 will lower blood pressure and prevent and treat hypertension. The inventors of the present invention have found such a substance having an angiotensin converting enzyme inhibitory activity in kiwi, and completed the present invention. More specifically, it has been confirmed that the extracts of the alopecia areata, especially the enzyme hydrolysates thereof, have a high angiotensin converting enzyme inhibitory activity.

In the present invention, the chitosan used as a component having an angiotensin converting enzyme inhibitory activity is a morphological variant, and its scientific name is Atrina pectinata , and its English name is pen-shell. It is an edible shellfish belonging to Phylum Mollusca, Bivalvia, Order Mytiloida, and Family Pinnidae taxonomically. It has a great clinical value because it contains proteins, essential amino acids, vitamins, minerals, tastes and flavors. Studies on these key shellfishes were limited to basic ecology and aquaculture research, and research on the search for physiologically active substances in key shellfish was insufficient. The present invention has revealed an angiotensin converting enzyme inhibitory activity by searching for a physiologically active substance of a Chinese cabbage. The keyhole according to the present invention means keyhole meat.

Accordingly, the present invention provides a pharmaceutical composition for inhibiting angiotensin converting enzyme and a health functional food, wherein the extract is an effective ingredient. The kiwi extract according to the present invention refers to a material having components of kiwi shell separated by a conventional extraction method in the field of the present invention.

The extract of Kjazaki used in the present invention can be extracted using an appropriate solvent such as water, methanol, ethanol, butanol, ethyl acetate, etc., which is used in a conventional solvent extraction method after pulverizing dried kjazaki, and an angiotensin converting enzyme inhibitory activity Any extraction method is irrelevant. However, preferably, water (more preferably, hot water) is used as an extraction solvent.

In addition, the present invention provides a pharmaceutical composition for inhibiting angiotensin converting enzyme and a health functional food, which comprises an enzyme hydrolyzate of Angelica keiskei koidz. As an active ingredient. Preferably, the enzymatic hydrolyzate of the keyhole is an extract or a purified product of the hydrolyzate of the keyhole enzyme, and the extraction or purification can be carried out by a conventional method. The enzymatic hydrolyzate of the keyhole according to the present invention is an enzyme hydrolyzate obtained by hydrolyzing a flesh of a chicken with a hydrolytic enzyme such as Alcalase (manufactured by Novo Co.), Flavorzyme (manufactured by Novo Co.), Protamex (Produced by Novo Co.), Nutrase (manufactured by Novo Co.), papain (manufactured by Sigma Co.), trypsin (manufactured by Sigma Co.), chymotrypsin (manufactured by Sigma Co.) Means a hydrolyzed material. The enzymatic hydrolyzate of the keychain has higher angiotensin converting enzyme inhibitory activity than the keychain extract.

The enzymatic hydrolysates of the chickshells exhibit different angiotensin converting enzyme inhibitory activities depending on the enzymes, but exhibit higher angiotensin converting enzyme inhibitory activity than the chrysanthemum extract. In particular, the hydrolyzate by protease is the most effective inhibitor. Angiotensin converting enzyme inhibitory activity is different depending on the type of enzymatic hydrolyzate of the key shellfish, because the type of amino acid located at the N-terminal and C-terminal is different from that of the enzyme, do. In addition, the enzymatic hydrolyzate of chimpanzee has higher angiotensin converting enzyme inhibitory activity than that of chimpanzee extract because it is decomposed by enzymatic hydrolysis to generate various peptides.

Therefore, when the pharmaceutical composition for inhibiting angiotensin converting enzyme according to the present invention or the health functional food is contained, the blood pressure lowering effect is obtained.

Also, the present invention provides a method for producing a recombinant yeast strain, comprising the steps of: (S1) adding a protease to a keyhole; (S2) heating the reaction solution after the step S1 to inactivate the proteolytic enzyme in the reaction solution; And (S3) filtering the supernatant obtained by centrifuging the reaction solution after the step (S2). The method for producing an enzyme hydrolyzate of angiosanno angiotensin converting enzyme having an angiotensin converting enzyme inhibitory activity is provided. At this time, the enzyme is preferably hydrolyzed using proteomes.

Also, the present invention provides a method for producing a recombinant yeast strain, comprising the steps of: (S1) adding a protease to a keyhole; (S2) heating the reaction solution after the step S1 to inactivate the proteolytic enzyme in the reaction solution; And (S3) a step of filtering the supernatant obtained by centrifuging the reaction solution after the step (S2), thereby improving the angiotensin converting enzyme inhibitory activity of the rat. At this time, the enzyme is preferably hydrolyzed using proteomes.

The present invention also provides a peptide for inhibiting angiotensin converting enzyme comprising a peptide consisting of Arg-Gln-Pro, preferably Arg-Gln-Pro. Arg represents an L-arginine residue, Gln represents an L-glutamine residue, and Pro represents L-proline.

The peptide competitively binds angiotensin-converting enzyme to angiotensin-converting enzyme to decrease the activity of angiotensin converting enzyme. Peptides showing inhibition of angiotensin converting enzyme activity are more influenced by the composition of the constituent peptides and the arrangement of amino acids than the composition and content of the constituent amino acids. In other words, it has an angiotensin converting enzyme inhibitory action by complex action such as peptide length, structure, kind of amino acid, arrangement order and the like. Therefore, the peptide according to the present invention can be used as a hypotensive agent.

The peptide can be obtained by chemical suitability but can be obtained through separation and purification of an enzyme hydrolyzate of chimpanzee having an angiotensin converting enzyme inhibitory activity. The separation and purification of the peptide may be carried out by various chromatographies such as the same techniques as those used for purification of oligopeptides, such as ion exchange chromatography, adsorption chromatography, reversed phase chromatography, partition chromatography and gel filtration chromatography , Solvent precipitation, salting out, and the like. Upon purification of the peptide of the present invention, the fraction containing the target substance can be separated and purified by determining the inhibitory action and / or yield of angiotensin converting enzyme as an indicator.

The peptidoglycan extract according to the present invention, the enzyme hydrolyzate of chimpanzee, or the peptide comprising Arg-Gln-Pro has an angiotensin converting enzyme inhibitory action and a bradykinin inactivation inhibitory action, resulting in a blood pressure lowering action. Accordingly, the pharmaceutical composition for inhibiting angiotensin converting enzyme comprising the above substances as an active ingredient and the health functional food can be used for various diseases derived from hypertension such as cerebral hemorrhage, cerebral infarction, angina pectoris, myocardial infarction, A prophylactic agent or a therapeutic agent, specifically, a hypotensive agent.

The pharmaceutical composition for inhibiting angiotensin converting enzyme according to the present invention may further comprise an inhibitor of angiotensin converting enzyme inhibitory action known in the art or a substance to be known in the future as well as a peptide conglomerate of Arg-Gln-Pro according to the present invention, And medicines may be used in combination.

In order to achieve the object of the present invention that the angiotensin converting enzyme inhibiting activity or the blood pressure lowering, the pharmaceutical composition for inhibiting angiotensin converting enzyme of the present invention may be administered at about 0.01 mg / kg to about 10 g / kg daily, mg / kg to about 1 g / kg per day. For convenience, the total daily dose may be divided as needed and divided into several doses throughout the day. However, the dosage may vary depending on the administration route and formulation, the patient's condition (age, sex, weight, etc.), the severity of the condition being treated, the purity and content of the enzyme hydrolyzate,

The active ingredient of the angiotensin converting enzyme inhibitor according to the present invention may be administered by any of oral administration or parenteral administration. Examples of the parenteral administration include intravenous injection and rectal administration.

The angiotensin converting enzyme inhibitor according to the present invention may be a solid preparation, a semi-solid preparation or a liquid preparation. Solid preparations include, but are not limited to, powders, granules, tablets, capsules, suppositories, and the like. Solid form preparations may include, but are not limited to, excipients, flavoring agents, binders, preservatives, disintegrants, lubricants, fillers, and the like. Examples of the semi-solid preparation include a cream, a lotion, an emulsifier, a liniment, and the like, but the present invention is not limited thereto, and can be produced by adding a suitable colorant, a flavoring agent, a stabilizer, a tackifier, a surfactant, and the like. Examples of the liquid preparation include water, alcohols, solutions such as solutions of propylene glycol, suspensions, emulsions and the like, but not limited thereto, and they can be prepared by adding appropriate coloring agents, flavoring agents, stabilizers, tackifiers and the like.

For example, powders can be prepared by simple mixing of the active ingredient of the present invention with suitable pharmaceutically acceptable excipients such as lactose, starch, microcrystalline cellulose and the like. The granules may contain the active ingredient of the present invention; Suitable excipients which are pharmaceutically acceptable; And a suitable pharmaceutically acceptable binder such as polyvinylpyrrolidone or hydroxypropylcellulose, followed by wet granulation using a solvent such as water, ethanol or isopropanol or dry granulation using a compressive force . Further, the tablet may be prepared by mixing the above granule with a suitable pharmaceutically acceptable salt such as magnesium stearate and then tableting it using a tablet machine.

The health food for inhibiting angiotensin converting enzyme according to the present invention can be used as a food additive, for example, an extract of Alaska pollack, an enzyme hydrolyzate of Alaska pollack or an peptide of Arg-Gln-Pro according to the present invention. For example, these materials may be added as they are, or may be used suitably in accordance with conventional methods such as by using them in combination with other foods or other food ingredients.

The mixing amount of the active ingredient for inhibiting angiotensin converting enzyme according to the present invention can be suitably determined according to the intended use (health or therapeutic treatment). Generally, the composition of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material, when the food or beverage is produced. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, it may be less than the above range, and since there is no problem in terms of stability, the active ingredient may be used in an amount exceeding the above range.

The kind of the food is not particularly limited. Examples of the food to which the active substance for inhibiting angiotensin converting enzyme according to the present invention can be added include meat products such as meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, Various soups, beverages, tea drinks, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as additional components such as ordinary beverages. Natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tau Martin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, there may be used various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, Carbonating agents, and the like. In addition, the substances according to the invention may contain flesh for the production of natural fruit juice, fruit juice drinks and vegetable drinks. These components may be used independently or in combination.

The enzymatic hydrolyzate of the extract according to the present invention has an angiotensin converting enzyme inhibitory activity. The peptides according to the present invention also have an angiotensin converting enzyme inhibitory activity. The substances having an angiotensin converting enzyme inhibitory activity are valuable as natural products derived from foods.

In addition, the substance having an angiotensin converting enzyme inhibitory activity according to the present invention is very useful for prevention and treatment of hypertension due to blood pressure lowering effect.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate exemplary embodiments of the invention and, together with the description of the invention, It should not be construed as limited.
Figure 1 is a mechanism involved in the renin-angiotensin system.
Fig. 2 is a view showing a method for producing an enzyme hydrolyzate of a keychain according to the present invention.
3 is a diagram showing a method for separating and purifying an angiotensin converting enzyme inhibitor peptide according to the present invention.
FIG. 4 is a graph showing the inhibitory activity of angiotensin converting enzyme of the enzyme hydrolyzate of the extract of the alginate extract and the alginate extract according to the present invention.
FIG. 5 is a graph showing the inhibitory activity of angiotensin converting enzyme according to the concentration of the hydrolyzate by the protease enzyme of the keyhole according to the present invention. FIG.
FIG. 6 is a graph showing the inhibitory activity and yield of angiotensin converting enzyme of five fractions obtained by separating protamex hydrolyzate of chimpanzee according to the present invention using an ultrafiltration membrane. FIG.
FIG. 7 is a chromatogram obtained by using an ODS AQ column of a low molecular peptide having a molecular weight of 1,000 dalton or less according to the present invention.
FIG. 8 is a graph showing the inhibition activity and yield of angiotensin converting enzyme of each fraction separated by ODS AQ column of a low-molecular peptide having a molecular weight of 1,000 dalton or less according to the present invention.
FIG. 9 is a chromatogram of PO3 according to the present invention obtained using a Vydac C-18 column. FIG.
10 is a graph showing the angiotensin converting enzyme inhibitory activity of each fraction of PO3 according to the present invention isolated by Vydac C-18 column.
11 is a chromatogram obtained by rechromatography of PO3V4 according to the present invention.
12 is a graph showing the angiotensin converting enzyme inhibition activity of each fraction separated by rechromathography of PO3V4 according to the present invention.
13 is a chromatogram obtained by gel permeation chromatography of PO3V5 according to the present invention on a Superdex Peptide HR column.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

≪ Preparation of Hot Water Extract of Kjoko "

Ingredients of chickpea meat

In July 2009, a natural shell (Atrina pectinata) from Chongsu Bay, Chungnam Province was sampled to remove the viscera and foreign matter, followed by lyophilization. It was stored frozen in powder form and used as an experimental sample.

Hot water extraction

Thirty times (w / w) of distilled water was added to the above-described kasha meat powder to homogenize it, and the mixture was subjected to hot-water extraction at 100 ° C for 4 hours in a heating mentle. The extract was centrifuged at 6,000 rpm for 15 minutes and the supernatant was taken. The supernatant was transferred to Advantec Filter Paar No. 2 (Toyo Roshi Kaisha, Ltd., Japan), and the filtered supernatant was lyophilized and pulverized to prepare a hot water extract of chitosan.

≪ Preparation of enzymatic hydrolyzate of pufferfish &

Enzymes used for hydrolysis

Alcalase 2.4 L, Flavourzyme 500 MG, Neutrase 0.8 L, and Protamex commercial protease were purchased from Novo Co. (Novozyme Nordisk, Bagsvaerd, Denmark) and used. Table 1 below shows the optimal reaction conditions for each enzyme and the composition of the enzyme.

enzyme Hydrolyzate  Produce

30% (w / w) of distilled water was added to the chicken meat powder, 1% of the protease was added thereto, and the reaction was allowed to proceed under optimal conditions for 2 hours. After the reaction, the mixture was heated at 90-100 ° C for 10 minutes to inactivate the enzyme. The supernatant was obtained by centrifugation at 6,000 rpm for 15 minutes. The supernatant was filtered with a 0.45 μm syringe filter. The filtered supernatant was lyophilized and powdered. The powder samples were stored at -20 ° C and used as analytical samples. The production method of the enzyme hydrolyzate is shown in Fig.

enzyme Optimum condition Composition of enzyme Temperature (℃) pH Alcalase 50 7.0 An endoprotease Neutrase 50 7.0 Containg both endoprotease and eptidase activities Flavourzyme 50 7.0 An endoprotease Protamex 50 7.0 A bacterial protease complex for the hydrolysis of food protein

<Measurement of Angiotensin Converting Enzyme Inhibitory Activity>

Angiotensin converting enzyme inhibitory activity of enzyme hydrolysates of hot water extracts and kinshoes were measured by Yamamoto et al. (1980). Angiotensin converting enzyme was prepared by adding 10 mL of 0.1 M sodium borate buffer (pH 8.3) to 1 g of the sample purified with acetone powder (Sigma, St Louis, MO, USA) in the lungs of rabbits and then stirring at 5 ° C for 24 hours. The supernatant obtained by separation at 4000 rpm for 30 minutes was used as an enzyme solution for angiotensin converting enzyme.

Angiotensin converting enzyme inhibition activity was determined by adding 100 μL of 0.1 M sodium borate buffer (pH 8.3) and 50 μL of angiotensin converting enzyme enzyme solution to 50 μL of the sample prepared at a concentration of 10 mg / mL, preincubating at 37 ° C. for 10 minutes, To 5 mL of sodium borate buffer (pH 8.3) containing 0.3 M NaCl, 50 μL of a substrate made by adding 25 mg of HHL (hippuryl-histidyl-leucine) was added and reacted at 37 ° C for 30 minutes. Then, 250 μL of 1 M HCl was added to stop the reaction. For the blank test, 50 μL of distilled water was used instead of the sample solution. In the control group, 250 μL of 1 M HCl was added first to stop the reaction, and then the enzyme solution was added. To this, 1.5 mL of ethyl acetate was added, stirred for 15 seconds, and centrifuged (3,000 rpm, 5 minutes, 4 ° C) to take 1 mL of the supernatant. The supernatant was dried at 120 ° C for 30 minutes, and then allowed to stand at room temperature for 5 minutes. Distilled water (3 mL) was added to dissolve for 15 seconds and the absorbance was measured at 228 nm. The inhibition rate of angiotensin converting enzyme , And the angiotensin converting enzyme inhibitory activity was calculated using the following formula.

)

ACE inhibition activity (%) = (

)

S: absorbance of the sample

C: Absorbance of blank test (group containing distilled water)

B: Absorbance of the control group (group in which the enzyme was added after stopping the reaction by HCl)

Statistical processing

Each experiment was statistically processed using the SAS (1999-2001) program. The mean and standard deviation of each group were calculated, and the difference between the samples was measured using Duncan's multiple rage test And analyzed the result difference between treatments.

<Isolation of Angiotensin Converting Enzyme Inhibiting Active Substance from Enzymatic Hydrolyzate of Chrysanthemum morifolium>

Angiotensin converting enzyme inhibition peptides were purified and purified by ultrafiltration, column chromatography and HPLC (AKTA explorer 100, pharmacia, Sweden) as shown in FIG. Detection of the peptide components was carried out at 215 nm using a UV detector mounted on HPLC.

Ultrafiltration

Ultrafiltration was performed according to the method of Tugeon et al. (1990) in order to remove the polypeptide of the polymer and to efficiently perform the separation process of the low molecular weight peptide. YM-1, YM-10, and YM-100 membranes with a molecular weight cut-off of 1,000 dalton and 3,000 dalton, 10,000 dalton, and 100,000 dalton, respectively, were subjected to DIAFLO ultrafiltration membrane (100,000 dalton, 100,000-10,000 dalton, 10,000-3,000 dalton, 3,000-1,000 dalton, 1,000 dalton or less), respectively, and then lyophilized using the above-mentioned method (Amicon Co., Beverly, MA, USA) Each of the angiotensin converting enzyme inhibitory effects was measured

Separation by ODS AQ column

Separation by ODS-AQ column was performed by modifying the method of Matsui et al. (1993). The ODS-AQ colum (26 ㅧ 300 mm, 0.25 g) was prepared by dissolving 0.2 g of the freeze-dried sample, which was excellent in inhibition of angiotensin converting enzyme inhibition, in 2 mL of distilled water for HPLC, Vt: 170 mL). At this time, the eluate was separated by gradient solvent using HPLC solvent A and HPLC solvent B as gradient. The eluate was eluted at a flow rate of 10 mL / min and 10 mL aliquots were collected. The fractions were detected at 215 nm and 280 nm, Were collected, concentrated under reduced pressure, and lyophilized in vacuo to measure angiotensin converting enzyme inhibitory activity.

Separation by Vydac C-18 column

Analysis of RP (reverse-phase) -HPLC by Vydac C-18 column was separated into two steps according to the solvent conditions.

Separation by linear gradient elution

The fractions of the peptides fractionated by the ODS-AQ column, which were excellent in the activity of inhibiting angiotensin converting enzyme, were dissolved in 2 mL of distilled water for HPLC to prepare a 10% solution, and the fractions were subjected to Vydac C-18 column (10 ㅧ 250 mm, Vt : 19.6 mL). At this time, the eluate was separated in gradient condition using 0.05% TFA in water as solvent A and 0.05% TFA in acetonitrile (ACN) as B solvent. The flow rate was 2 mL / min and eluted with 2 mL each. And each fraction was collected and concentrated under reduced pressure, and then freeze-dried in vacuo to measure angiotensin converting enzyme inhibitory effect.

Isocratic elution

The fraction eluted first with the Vydac C-18 column (10 ㅧ 250 mm, Vt: 19.6 mL) was re-injected into the Vydac C-18 column with 0.07% TFA And 3% acetonitrile (ACN) in water. The eluate was eluted at a flow rate of 2 mL / min, and 2 mL aliquots were collected. The fractions were detected at 215 nm and 280 nm, and the fractions were collected After concentration under reduced pressure, the cells were lyophilized in vacuo and the inhibitory effect of angiotensin converting enzyme was measured.

Isolation by Superdex Peptide HR column

The peptide fractions fractionated by Vydac C-18 column were injected into a Superdex peptide column (10 ㅧ 300 mm, Vt: 24 mL). The eluate was eluted at a flow rate of 0.5 mL / min with HPLC water, The fractions were collected at 215 nm and 280 nm, respectively, and the fractions were collected, concentrated under reduced pressure, and lyophilized in vacuo to remove impurities of the finally remaining salts and purified.

<Amino acid sequence analysis of peptide>

The amino acid sequences of the purified and purified amino acid sequences of the angiotensin converting enzyme inhibiting peptide fragments were analyzed by Superdex peptide HR column chromatography using Procise ™ (Perkin Elmer, protein sequencing system, Foster, CA, USA) And the standard was measured. Then, the sample was analyzed using 20 L of sample.

<Evaluation of inhibitory effect of angiotensin converting enzyme on hydrolysates of enzymes hydrolysates>

In order to investigate the antihypertensive activity of hydrolyzed samples by Alcalase, Flavourzyme, Protamex, and Neutrase, which are commercial enzymes that mainly use chickpea meat, the results of measuring angiotensin converting enzyme inhibitory activity at a sample concentration of 10 mg / 4. No enzyme (hydrothermal extract) showed better inhibitory activity of enzyme hydrolysates. Angiotensin - converting enzyme inhibitory activity of protamex was 85.7% and angelolytic enzyme activity of Flavourzyme and Neutrase was 56.5% and 52.6%, respectively. In the case of using Alcalase, the activity was 47.4%, which was lower than other enzymes.

Angiotensin converting enzyme inhibition activity of protamex hydrolyzate was investigated. As shown in FIG. 5, the inhibition effect of angiotensin converting enzyme was increased in a concentration dependent manner as the sample concentration increased. Enzymatic hydrolysates of Kjazuki may inhibit angiotensin - converting enzyme action and may be effective in preventing hypertension.

<Structural Analysis of Angiotensin Converting Enzyme Inhibitory Peptide>

Selection of enzyme hydrolysates

In the above results, Protamex hydrolyzate, which has the highest inhibitory effect on angiotensin converting enzyme, was selected as a sample in the next experiment, which was hydrolyzed by Alcalase, Flavourwyme, Protamex and Neutrase. &Lt; / RTI &gt;

Angiotensin converting enzyme inhibitory activity according to molecular weight

Using the ultrafiltration membrane, the inhibitory activity and yield of angiotensin converting enzyme of 5 fractions are shown in FIG. The inhibition effect of angiotensin converting enzyme was highest in 62.9% of the fraction with molecular weight of 1,000 dalton or less, 61.9% between 1,000-3,000 dalton, 57.2% between 3,000-10,000 dalton, 28.0% between 10,000-100,000 dalton and more than 100,000 dalton 12.2%, the angiotensin converting enzyme inhibition activity was higher as the molecular weight was lower, indicating that the angiotensin converting enzyme inhibitor peptide of the enzyme hydrolyzate of chrysanthemum morifolium was also low molecular weight.

The yields of the fractions having a molecular weight of less than 1,000 dalton accounted for 69.2%, 11.6% for 1,000-3,000 dalton, 9.1% for 3,000-10,000 dalton, 8.3% for 10,000-100,000 dalton and 7.5% for fractions over 100,000 dalton Respectively.

 Therefore, considering the yield and inhibition effect of angiotensin converting enzyme, the molecular weight of less than 1,000 dalton (Fraction 1) was selected as the next separation step.

Separation of Angiotensin Converting Enzyme Inhibitory Peptides by ODS AQ Column

After lyophilization of low molecular weight peptide (Fraction 1) with molecular weight less than 1,000 dalton and high yield of enzyme hydrolysates obtained by ultrafiltration and high yield of inhibition of angiotensin converting enzyme, ODS AQ which can perform salt removal and peptide fractionation simultaneously The chromatogram of FIG. 7 was obtained using one column. The chromatogram was divided into four fractions (PO1, PO2, PO3, PO4) by elution with an elution volume of 340 mL and an ethanol concentration of 0 to 60% solution. The absorbance at which the amount of peptide can be measured simply by causing refraction of light at the peptide binding site was measured at 215 nm, and the activity and yield of inhibition of angiotensin converting enzyme by the four fractions were measured and shown in FIG. PO1, PO2, PO3 and PO4 inhibited angiotensin converting enzyme by 16.9%, 53.1%, 70.4% and 73.4% respectively and the yields of PO1, PO2, PO3 and PO4 were 51.3%, 24.1%, 16.8% and 7.7% Respectively.

The fraction (PO3) eluted with 20-40% ethanol solution was selected as the next separation step by considering the inhibitory activity and yield of angiotensin converting enzyme.

 Isolation of angiotensin converting enzyme inhibitor peptide by Vydac C-18 column

Separation by linear gradient elution

The fraction (PO3) selected as a sample in the ODS-AQ column chromatography was lyophilized, and then the chromatogram of FIG. 9 was obtained using a Vydac C-18 column. This chromatogram was divided into four fractions (PO3V1, PO3V2, PO3V3, PO3V4) eluted with an ACN solution containing 45 mL of elution volume and 0.05% TFA. The absorbance at which the amount of peptide can be measured simply by causing refraction of light at the peptide binding site was measured at 215 nm, and the activity of inhibiting angiotensin converting enzyme in the four fractions was measured and shown in FIG. PO3V1, PO3V2, PO3V3 and PO3V4 showed angiotensin converting enzyme inhibitory activity of 3.6%, 7.6%, 2.5% and 27.4%, respectively.

The fraction (PO3V4) was selected as the next separation step in consideration of the inhibitory effect of angiotensin converting enzyme among the four fractions.

Isocratic elution

In general column chromatography, pH and mobile phase of solvent are important factors for fractionation (Jeong et al., 2003). In this experiment, the fraction eluted with ACN solution containing 0.05% TFA was subjected to rechromatography by changing the mobile phase after lyophilization of PO3V4 having high angiotensin converting enzyme-inhibiting activity. As a result, it was separated into five fractions as shown in Fig. Fig. 12 shows the angiotensin converting enzyme inhibitory activity of PO3V4V1, PO3V4V2, PO3V4V3, PO3V4V4 and PO3V4V5 at 4.7%, 4.2%, 5.2%, 5.5% and 10.8%, respectively. Among them, the fraction (PO3V4V5) was selected as the next separation step in consideration of the angiotensin converting enzyme inhibitory effect.

Separation of Angiotensin Converting Enzyme Inhibitory Peptides by Superdex Peptide HR Column

The final separation of the angiotensin converting enzyme inhibitor peptide by Vydac C-18 column chromatography was performed by HR column chromatography to remove impurities such as salts finally remaining in the final separated supernatant, and to confirm the degree of each purification, Superdex Peptide HR column was subjected to gel permeation chromatography to obtain the chromatogram of FIG. A single peptide (PO3V4V5S) was obtained with a Superdex Peptide HR column. These results indicate that the peptides having high angiotensin inhibitory activity have been sufficiently purified through the first and second purification steps and the purified fractions are purified into a single peptide in the third purification step. Finally, a single peptide (PO3V4V5S) was used as a sample for amino acid sequence measurement.

The amino acid sequence of the angiotensin converting enzyme inhibitor peptide

The amino acid sequence of the final isolated angiotensin converting enzyme inhibitory peptide, single fraction (PO3V45S) was analyzed by Superdex Peptide HR column chromatography. PO3V4V5S is a peptide consisting of three amino acids. The amino acid sequence is shown in the order of Arg-Gln-Pro. The molecular weight of this peptide is calculated to be 435.3 dalton.

Based on the results of the experiment, it was confirmed that the inhibitory action of the substance isolated from the keychain was more influenced by the type of the constituent peptide or the amino acid sequence than the composition and content of the constituent amino acid. In other words, the inhibition of angiotensin converting enzyme by the substance isolated from the keychain is presumed to act on the inhibition of angiotensin converting enzyme by a complex action such as the length, structure, amino acid type and sequence order of the low molecular peptide.

Claims (15)

A pharmaceutical composition for lowering blood pressure for inhibiting angiotensin-converting enzyme (ACE), which comprises an enzymatic hydrolyzate of chinchillas as an active ingredient. The pharmaceutical composition according to claim 1, wherein the enzyme is Protamex. [Claim 2] The pharmaceutical composition according to claim 1, wherein the enzyme hydrolyzate of the keychain is an extract or a purified product of an enzyme hydrolyzate of a Chinese cabbage. delete A health functional food for inhibiting angiotensin-converting enzyme (ACE) inhibiting blood pressure, characterized by containing an enzyme hydrolyzate of a Chinese cabbage as an active ingredient. [Claim 6] The health food according to claim 5, wherein the enzyme is Protamex. [Claim 7] The health food according to claim 5, wherein the enzymatic hydrolyzate of the keychain is an extract or a purified product of an enzyme hydrolyzate of anchovy. delete (S1) adding a protease to a keyhole;
(S2) heating the reaction solution obtained in step S2 to inactivate the proteolytic enzyme in the reaction solution; And
(S3) A step of filtering the supernatant obtained by centrifuging the reaction solution obtained in the step S2
Wherein the angiotensin converting enzyme inhibitory activity is increased. 10. The method of claim 9, wherein the enzyme is Protamex. Arg-Gln-Pro as an active ingredient. The pharmaceutical composition for inhibiting angiotensin converting enzyme according to claim 1, wherein the peptide is selected from the group consisting of Arg-Gln-Pro and Arg-Gln-Pro. delete Arg-Gln-Pro as an active ingredient. The present invention also provides a health functional food for inhibiting angiotensin converting enzyme. delete Arg-Gln-Pro.

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